Detection and characterization of multidrug resistant Escherichia coli carrying virulence gene isolated from broilers in Bangladesh

Abstract Background The emergence and dissemination of multidrug resistant (MDR) bacteria pose a severe threat to public health by limiting clinical treatment and prophylactic options. Objectives This study investigates the prevalence of Escherichia coli in broilers, their phenotypic antimicrobial resistance (AMR) profiles and the presence of virulence‐associated genes (VAGs) and antimicrobial resistance genes (ARGs) using polymerase chain reaction (PCR). Materials and methods A total of 216 pooled cloacal samples were collected from 1080 broilers across six districts of Bangladesh. Each pooled sample comprised randomly selected cloacal swabs from five birds per farm. E. coli isolates were identified using standard bacteriological approach, followed by biochemical assays and PCR. Antimicrobial susceptibility was assessed using the Kirby–Bauer disc diffusion method, and the presence of ARGs and VAGs was determined via PCR. Five selected isolates were partially sequenced for five VAGs using Sanger sequencing. Results A total of 177 E. coli isolates (81.94%, 95% confidence interval: 76.24%–86.53%) were identified. The isolates showed the highest resistance to ampicillin (93.79%), followed by tetracycline (91.53%), erythromycin (89.27%) and ciprofloxacin (87%). Conversely, ceftriaxone (80.79%) showed highest susceptibility, followed by gentamicin (37.29%) and neomycin (31.07%). All isolates were MDR, with a multiple antibiotic resistance indexes were <0.3. A significant percentage (16.38%) of E. coli isolates were MDR to five antimicrobial classes and harboured bla TEM, sul1, ere (A), tetA, tetB and tetC genes. The highest prevalent ARGs were bla TEM (88.14%) followed by ere (A) (83.62%) and sul 1 (72.32%). The prevalence of VAGs was astA (56.50%), iucD (31.07%), iss (21.47%), irp2 (15.82%) and cva/cvi (3.39%), respectively. Conclusions This study highlights the presence of ARGs contributing to the development of MDR in E. coli carrying VAGs in broilers. Effective monitoring and surveillance of antimicrobial usage in poultry production systems are urgently required to prevent emergence and dissemination of AMR.


INTRODUCTION
Escherichia coli, which has been considered a harmless and versatile microbe, can lead to various intestinal and extra-intestinal diseases, including haemorrhagic diarrhoea, urinary tract infections, meningitis and pneumonia, in humans and animals by acquiring different virulence traits (Hammerum & Heuer, 2009;Kunert Filho et al., 2015;Parvin et al., 2020).Commonly used for treating and preventing diseases in poultry, antimicrobials were used for growth promotion in poultry production, leading to antimicrobial misuse (Hassan et al., 2021).Bacteria have acquired drug resistance genes through horizontal gene transfer or gene mutation mechanisms (Darby et al., 2023).
The poor selection and irrational use of antimicrobials are the leading causes of the emergence and dissemination of antimicrobial resistant bacteria, which can potentially be transmitted to humans through the food chain (Hasib et al., 2024;Parvin et al., 2020).According to the World Health Organization (WHO), millions of patients are expected to die in the coming decades due to antimicrobial resistance (AMR), as antimicrobials fail to treat clinical cases of infections (WHO, 2023).Bangladesh, a major poultry producer, according to the Department of Livestock Service Economy 2019-2020, relies on the poultry industry to drive economic growth in rural communities.In this context, approximately 3563.18 lakhs of poultry were generated from a total livestock production of 4122.44 lakhs (DLS, 2020).The extent of antimicrobial usage (AMU) in poultry and the annual sales data of antimicrobials in Bangladesh remain unknown (Imam et al., 2020).
Farmers can easily acquire antimicrobials from drug sellers without needing a prescription from a registered veterinarian (Ahmed et al., 2020).Despite recent stringent regulations on antimicrobial sales, unregulated use of antimicrobials is widespread in poultry production in Bangladesh (Hassan et al., 2021).
Multidrug resistance has been increased all over the world that is considered a public health threat.Several recent investigations reported the emergence of multidrug resistant (MDR) bacterial pathogens from different origins that increase the necessity of the proper use of antibiotics.Besides, the routine application of the antimicrobial susceptibility testing (AST) to detect the antibiotic of choice as well as the screening of the emerging MDR strains (Algammal et al., 2019(Algammal et al., , 2022(Algammal et al., , 2023;;Elbehiry et al., 2022;Shafiq et al., 2022).
Pathogenicity is expedited by virulence factors like adhesion, iron acquisition, haemolysin, aerobactin's and serum resistance translated by virulence-associated genes (VAGs) found in plasmids or chromosomal areas of pathogenicity islands (PAIs) (Subedi et al., 2018).These virulence factors permeate host tissues, produce toxins, evade host defences and ultimately cause local inflammation in the host, among other pathways that lead to infection (El-Baz et al., 2022).Isolates with at least five virulence genes are classified as avian pathogenic E. coli (APEC), and five genes (iutA, iss, ompT, iroN and hlyF) mainly were associated with pathogenesis (De Carli et al., 2015).
E. coli also causes colibacillosis, resulting in considerable disease in the poultry industry worldwide (Kika et al., 2023).The impacts include production losses, increased mortality and higher production costs due to systemic infections in the poultry gut, characterized by acute fatal septicemia or sub-acute fibrinous pericarditis, air sacculitis, salpingitis and peritonitis (Ibrahim et al., 2019).Moreover, the presence of antimicrobial resistance genes (ARGs) and VAGs in the farm makes it more vulnerable to contaminating the surrounding environment and adds selection pressure on the environmental pathogens.Maintaining effective biosecurity on broiler farms appears to be impractical with current farming practices in Bangladesh, which would exacerbate the situation in terms of potential zoonotic pathogen transmission to humans and other animals.
AMR is a burning issue, and there is a lack of stringent strategies to control AMU in poultry production in Bangladesh.Given the importance of AMR and VAGs in E. coli, this study was undertaken to estimate the prevalence of E. coli from cloacal samples of broiler chickens.Subsequently, the phenotypic AMR profile was examined, followed by molecular identification of ARGs and VAGs to establish a baseline of AMR patterns and VAGs found in E. coli isolated from broiler chickens in Bangladesh.

Sampling
F I G U R E 1 A map of Bangladesh displaying the geographical location of broiler farms included in the study. calculation: where n is the sample size, N is the population size, critical value (95% level of significance) Z is the 1.96 s, margin of error e is the 0.05 and sample proportion p is the 0.5.
Later, one pooled cloacal swab sample was collected for each of the representative farms from five randomly selected birds from the broiler flocks.Swabs were taken aseptically using commercial swabs (Model: PW005 Himedia) and transferred into the falcon tube containing 5 mL buffered peptone water (BPW) (Oxoid Ltd.).All samples were stored at 4 • C and transferred carefully for laboratory processing (not more than 24 h) and subsequent isolation.

Isolation and identification of E. coli
Isolation and identification were performed following standard bacteriological methods (Dutta et al., 2020).Briefly, all samples were pre-enriched overnight in BPW (Oxoid Ltd.).A loopful broth (∼10 μL) was inoculated onto MacConkey agar and eosin-methylene blue (EMB) agar (Oxoid Ltd.) and incubated overnight at 37 • C. The purified colonies were sub-cultured onto blood agar with 5% bovine blood (Oxoid Ltd.) and incubated overnight at 37

Molecular confirmation of E. coli
The genomic DNA was extracted following the conventional crude boiling method (Malorny et al., 2003).A polymerase chain reaction (PCR) assay was conducted for the final confirmation of the suspected isolates using a genus-specific universal primer targeting the 16S rRNA gene: (F) 5′-GACCTCGGTTTAGTTCACAGA-3′ and (R) 5′-CACACGCTGACGCTGACCA-3′, maintaining the initial denaturation at 95 • C for 5 min and final extension at 72 • C for 7 min with the for 1 min and extension at 72 • C for 1 min (Schippa et al., 2010).
The PCR products were visualized on a gel documentation system (UVP UVsolo touch-Analytik Jena AG, Thermo Fisher Scientific) after electrophoresis with a 1.5% agarose gel (MP Biomedicals).

Detection of AMR genes (ARGs)
Although phenotypically eight antimicrobials were tested in this study, the following ARGs were screened from the isolates -aminoglycoside resistant [aac(3)-IV], sulphonamide resistant (sul1), penicillin resistant (bla SHV , bla CMY , bla TEM ), erythromycin resistant [ere (A)] and TE resistant (tetA, tetB, tetC) by PCR.The oligonucleotide primers sequence and cyclic conditions used for each gene are shown in Table 1.

Detection of virulence-associated genes (VAGs)
All confirmed E. coli isolates were further investigated for targeted VAGs by multiplex PCR (Ewers et al., 2007).The primer sequences used for the PCR confirmation are summarized in Table 2.The reaction volume mixture was 25 μL containing 12.5 μL Taq 2X master mix (New England Biolabs Inc.), 1 μL forward and reverse primer (20 pmol/μL), 2 μL template DNA (average 3.35 ng/μL DNA) and 8.5 μL nucleasefree water.PCR was run on a thermocycler (Applied Biosystem, 2720 thermal cycler) following the cycling conditions: the initial denaturation at 94 • C for 3 min and final extension at 72 • C for 10 min with the 35 cycles of denaturation at 94 • C for 30 s, annealing at 58 • C for 30 s and extension at 68 • C for 3 min.

2.7
Sequencing of virulence-associated genes (VAGs) Five VAGs (astA, iss, irp2, iucD and cvi/cvaC) from five E. coli isolates were randomly selected for partial sequencing.The PCR products were purified using a DNA purification kit (Favorgen Biotech Corp.) and sequenced both forward and reverse strands by sanger method using a commercial service (Macrogen Inc.).The raw forward and reverse sequence read data were manually cleaned up with a chromatogram and assembled by CAP3 sequence alignment programme (Huang & Madan, 1999) to make consensus sequence.The consensus sequences were then deposited in the NCBI GenBank database for accession numbers (Accession numbers: MT928164-MT928166, MT982360-MT982361).

Statistical analysis
Data retrieved from the samples were inserted in the Microsoft Office Excel 2016 Excel sheet.The frequency (n), prevalence (%) and 95% confidence intervals (CIs) were calculated using the modified Wald method in the GraphPad software QuickCalcs (https://www.graphpad.com/quickcalcs/).The map and the farm's location were created using QGIS 3.12.0.The correlation coefficient between phenotypic and genotypic resistance was calculated using R software (version 4.4.1;https://www.r-project.org/).

RESULTS
AMR and VAGs in E. coli are major public health concerns worldwide, particularly in countries like Bangladesh, where poultry farming is popular.This study sought to assess the AMR patterns and VAG profiles of E. coli isolated from broiler cloacal samples in Bangladesh.
This study hypothesized that E. coli isolated from broiler would be MDR and have several VAGs, increasing their harmful potential.Thus, we collected cloacal swabs from broilers on farms in various regions of Bangladesh, isolated E. coli and assessed antibiotic susceptibility and VAGs using PCR.

Phenotypic characteristics and prevalence of recovered E. coli isolates
All E. coli isolates recovered in this study were Gram-negative, motile and rod shaped bacilli.The colonies were large, pink, lactose fermentative on MacConkey agar, showed metallic sheen on EMB agar and were hemolytic on blood agar.Additionally, all the isolates tested positive for catalase, lactose fermentation, indole and methyl red tests.Conversely, they tested negative for oxidase, H 2 S production and Voges-Proskauer test.A total of 216 pooled broiler cloacal samples were investigated.Among them, 177 of 216 samples (81.94%; 95% CI 76.24-86.53)were confirmed by PCR to be colonized with E. coli (Figure 2).

TA B L E 1
The oligonucleotide primer sequences for detection of antimicrobial resistance genes of Escherichia coli.
Moreover, the MAR index value in this study was >0.3, which indicated that recovered E. coli strains originated from high risk contamination.Furthermore, the correlation coefficient among phenotypic AMR and ARGs were estimated and revealed positive correlation between sul1 gene and SXT (r = 0.7), aac(3)-IV gene and CN (r = 0.8), ere (A) gene and E (r = 0.8), tet A gene and TE (r = 0.6), bla TEM gene and AMP (r = 0.7), bla SHV gene and CRO (r = 0.5) and bla CMY gene and CRO (r = 0.6), as illustrated in Figure 5.

No
The heatmap represents the correlation between antimicrobial agents and antimicrobial resistance genes (ARGs).

Distribution of VAGs
All the E. coli isolates (n = 177) were tested for the presence of VAGs of APEC.A total of eight virulence genes were tested in this study.

DISCUSSION
The comprehensive examination of E. coli in broiler cloacal samples conducted in this study sheds light on the complex interplay among poultry health, AMR and virulence factors.The phenotypic characteristics of recovered E. coli isolates were similar to a previously reported study by Algammal et al. (2022).In addition, the prevalence of E. coli in this study was 81.94%, which is higher than previous studies reported from Bangladesh (Dutta et al., 2020;Hossain et al., 2008; Sarker These resistance patterns align with the previous studies (Al Azad et al., 2019;Das et al., 2021;Subedi et al., 2018;Sarker et al., 2019).However, Al Azad et al. (2019) reported 100% resistance to ampicillin, tetracycline, erythromycin, ciprofloxacin and sulfamethoxazole/trimethoprim.
The high resistance level of these antimicrobials in broilers reflects the indiscriminate use of antimicrobials in broiler production by farmers in Bangladesh (Hassan et al., 2021).In contrast, ceftriaxone showed lower resistance (19%), which is expected for this type of cephalosporin as it is not used in broiler farms of Bangladesh (Hasan et al., 2011;Imam et al., 2020).
Furthermore, nine ARGs were screened and their prevalence was similar to a previous study from Bangladesh (Al Azad et al., 2019) but higher than a study reported from Iran (Momtaz et al., 2012).All the E. coli isolates in this study were MDR.However, this study could not classify the isolates as extensively drug resistant (XDR) or pandrug resistant (PDR) because only a few antimicrobials were tested.
According to Magiorakos et al. (2012), an adequate number of antimicrobials must be tested for Enterobacteriaceae to classify XDR and PDR.
Regarding the MDR patterns, the highest percentage of isolates were resistant to five or six antimicrobial classes and carried all ARGs tested in this study.It represents the misuse of antimicrobials in the poultry sector of Bangladesh.Resistance can spread through conjugation, transformation and transduction processes, making it possible for highly resistant isolates from broiler farms to transfer resistance markers to other bacteria.Apart from these, resistance is associated with other processes, including growth in the stationary phase, biofilms and persistence (Amer et al., 2018).This transfer occurs to humans through the food chain, mainly by consuming inadequately cooked meat or handling meat unsafely during processing (Nath et al., 2023) and is widely circulated to the environment through poultry waste (Elbehiry et al., 2022).Al Sattar et al. (2023) indicated that increasing farmers' awareness of antimicrobials has been improved AMR conditions in the farming environment.
In this study, PCR analysis revealed that most of the isolates carried varying amounts of VAGs.The most prevalent VAG identified was astA (56.5%), commonly found in EAEC pathotypes (Sidhu et al., 2013).
The genes iucD and irp2, related to iron acquisition, were present at frequency of 31.07%and 15.82%, respectively.This aligns with findings from Subedi et al. (2018), reported higher frequency of iucD (97.3%) compared to irp2 gene (73.3%) in Nepal.Additionally, the serum survival gene (iss) and colicin V plasmid operon gene (cva/cvi) were detected at frequencies of 21.47% and 3.39%, respectively.In contrast, papC, tsh and vat were not detected among the E. coli isolates.
The overall frequency of VAGs in this study was lower compared to reports from Nepal (Subedi et al., 2018), Jordan (Ibrahim et al., 2019) and Korea (Kwon et al., 2008).Although no isolates in this study were APEC, but most of the isolates carried two to three VAGs.These genes, often chromosomal or plasmid-mediated and integrated into PAI, cause colibacillosis (De Carli et al., 2015).They contribute to tissue damage, colonization and inflammatory reactions within the host, allowing pathogens to evade host defences, thus escalating the pathogenesis of MDR E. coli, thereby limiting treatment options (El-Baz et al., 2022;Shafiq et al., 2022).
The acquisition of VAGs usually occurs through horizontal gene transfer (Hacker & Kaper, 2000;Ochman et al., 2000), which may explain the absence or the low prevalence of certain VAGs in this study.The presence of virulence markers in E. coli is genetically similar with different pathogenic clones, indicating their potential for transferring virulence genes to pathogenic E. coli clones in humans and animals (Ahmed et al., 2020).However, several studies have taken an "integrated poultry production" approach and suggested that broiler breeders and hatcheries may be significant reservoirs of early APEC infections via environmental contamination or vertical transmission (Giovanardi et al., 2005;Petersen et al., 2006).
This study highlights the urgency of addressing resistance and virulence in poultry, emphasizing the significance of policy changes, strong legislation, law enforcement and collaborative efforts.By aligning with global standards and research-driven strategies, the poultry industry can effectively combat AMR, protect poultry health and mitigate potential public health risks.
Further studies on AMR using all listed and available antimicrobials for E. coli are recommended to understand the current status of AMR.
Additionally, investigations using the one health approach are urgently needed in Bangladesh to identify the spread of AMR pathogens, including those affecting clinical patients.A holistic farm-to-fork approach would be valuable for future research to identify contamination points in meat and assess consumer exposure to AMR pathogens by identifying potential hazards along the food chain.Moreover, additional studies on biofilm characteristics should be conducted to assess the potential pathogenicity of the isolates.

CONCLUSION
This study investigated the occurrence of VAGs in E. coli and identified their AMR patterns.The widespread AMR of E. coli and the identification of ARGs highlighted the importance of monitoring the spread of ARGs in poultry farms and in the environment.The presence of VAGs in MDR E. coli limits the treatment options for pathogenic strains.
Routine surveillance, monitoring and stakeholder awareness should be strengthened to prevent the dissemination of MDR E. coli as well as minimize possible risks to public health.
Oligonucleotide primer sequences for detection of target virulence genes of Escherichia coli isolates.
papC, tsh and vat were not detected among E. coli isolates.The VAG profile is presented in Table5 and Figure 7.
TA B L E 4 Distribution of antimicrobial resistance genes (ARGs) (n = 177) in Escherichia coli isolates.
TA B L E 5F I G U R E 7 Result of the polymerase chain reaction (PCR) assay for the virulence-associated genes of Escherichia coli isolates tested; Lane L: 1 kb plus DNA ladder; Lane N: negative control; Lane 1-5: samples.